Chemotaxis by the RAW264 mouse macrophage cell line was inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin. A search for biochemical reactions inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin has revealed that only one reaction, the synthesis of a small number of proteins identified after separation by two-dimensional polyacrylamide gel electrophoresis, has the necessary specificity for involvement in the 3-deazaadenosine-sensitive step of chemotaxis. A study with several adenosine analogs showed a correlation between inhibition of chemotaxis and inhibition of the synthesis of a common subset of proteins. These analogs also inhibited the synthesis of polyadenylated mRNA, leading us to postulate that incubation of cells with 3-deazaadenosine inhibits a methylation reaction that is required for the formation of a functional mRNA coding for one or more proteins required for chemotaxis. Experiments to identify attractant-specific proteins have been limited because chemically defined attractants for RAW264 cells have not been available. This problem has been overcome by the isolation of a stable cell hybrid from a fusion between human leukocytes and a thioguanine-resistant RAW264 cell line. The hybrid expressed functional genes for chemotaxis to fMet-leu-phe, a commercially available synthetic attractant. Binding of fMet-leu-phe to hybrid cell membranes indicated that the binding constant was 2 nM and each cell had an average of 1200 receptors. In addition to chemotactic receptors, one or more guanine nucleotide binding proteins are required for chemotaxis by RAW264 and the hybrid cells. This conclusion is based on the observation that chemotaxis of either RAW264 or hybrid cells is inhibited upon incubation of the cells with either cholera toxin or pertussis toxin. In all cases entry of the toxin is required and there is a correlation between toxin-catalysed ADP-ribosylation of a guanine nucleotide binding protein and the inhibition of chemotaxis. Although both cholera toxin and pertussis toxin affect cAMP levels, elevated cAMP levels per se do not inhibit chemotaxis.